Clinical Validation of a Prenatal Cell-Free DNA Screening Test for Fetal RHD in a Large U.S. Cohort

Abstract

OBJECTIVE: To present a large U.S. clinical validation of a next-generation sequencing–based, noninvasive prenatal cell-free DNA test for fetal RHD. METHODS: This clinical validation study assessed the performance of a commercially available, next-generation sequencing–based cell-free DNA test for fetal RHD status. Samples that passed quality metrics were included if the patient had a previously reported cell-free DNA result for fetal aneuploidy, maternal RhD-negative serology, newborn RhD serology, and maternal RHD deletion or RHD-CE-D hybrid(r's) genotype. Dizygotic twin pregnancies were excluded. Maternal and fetal RHD genotypes were evaluated with prospective cell-free DNA next-generation sequencing analysis. At the time of analysis, investigators were blinded to fetal RhD status. RESULTS: The cohort consisted of 655 pregnant patients with serologic results for RhD antigen. Patient demographics included a representative distribution of race and ethnicities in the RhD-negative U.S. population (74.0% White, 13.7% Hispanic, 7.0% Black, and 2.1% Asian). Cell-free DNA fetal RHD was not reported in two cases. There were zero false-negative cases; 356 of 356 fetuses were correctly identified as fetal RhD positive (sensitivity 100%, 95% CI, 98.9–100%). Of the 297 RhD-negative fetuses, 295 were correctly identified as RhD negative (specificity 99.3%, 95% CI, 97.6–99.8%). Of the fetuses with a negative RhD phenotype, the cell-free DNA test accurately identified three with the fetal RHD pseudogene (RHDΨ) genotype. CONCLUSION: Validation of this test in this large U.S. cohort of RhD-negative patients provides data on early and accurate noninvasive prenatal identification of fetal RHD genotype at 9 weeks of gestation or more. This test has the potential to assist patients and clinicians in the prevention and management of RhD alloimmunization.